Carbon Dioxide Test Procedures
Proprietors of bonded wine cellars,
taxpaid wine bottling houses,
and others concerned:
The purpose of this circular is to announce the addition of the enzymatic method to the
authorized test procedures for determining the amount of carbon dioxide added to or retained
in still wines under the provisions of 26 CFR 240.531 and 240.534. This method will be
published as an ATF Procedure in an early issue of the Alcohol, Tobacco and Firearms Bulletin.
Public Law 85-859 amended 26 U.S.C. 5041(a) to provide that a limited quantity of carbon
dioxide may be added to or retained in still wines. 26 CFR 240.534 provides that the Director,
Bureau of Alcohol, Tobacco and Firearms, may prescribe procedures for determining the carbon
dioxide content of still wines. Revenue Procedure 59-33 authorized two procedures, the
manometric method and the volumetric method (referred to by industry circular 59-47 as the
"Gasometric Procedures" and "Titrimetric Procedure").
The enzymatic method is hereby authorized by the Director as an acceptable test
procedure for the determination of the quantity of carbon dioxide added to or retained in
still wines. This method is described in detail below. Any of these three procedures,
the enzymatic method, the manometric method or the volumetric method, may be used to
determine the carbon dioxide content of still wines.
The Enzymatic Method
Preparation of reagent:
Carbonic anhydrase solution. Prepare aqueous solution containing approximately
1 milligram enzyme/milliliter. This solution is stable approximately 2 weeks in
Determination of Carbon Dioxide:
Cool sample to 0°C or less, so that it can be pipetted without loss of CO2. Gently
mix by inverting bottle several times. With automatic 25 or 30 milliliter pipet with
Teflon stopcock, dispense aliquot of 0.1N NaOH into beaker. Rinse 20 milliliter pipet
with sample to prevent warming sample with possible loss of CO2. Pipet sample with tip
submerged just below surface of NaOH in beaker. Add 3-4 drops enzyme, and place beaker
under glass and calomel electrodes. (Beckman 41263 and 40463 are satisfactory.)
Titrate to pH 8.45 with 0.1N H2SO4 from 5 milliliter buret graduated in 0.01 milliliter.
To correct for presence of acids other than H2CO3, place 50 milliliters wine
in 500 milliliter heavy-walled flask at room temperature and agitate 1 minute under
vacuum of approximately 27". Titrate 20 milliliters to pH 7.75 with 0.1N NaOH as
above. Subtract milliliters used from that used in first titration. Calculate as
follows: [(Net milliliters NaOH x normality) (milliliters H2SO4 x normality)]
x 100 x 44/milliliters sample = milligrams CO2/100 milliliters wine.
This procedure and the previously authorized procedures, except for minor editorial
changes, are the same as those appearing on pages 189-191 of the Eleventh Edition of
the Official Methods of Analysis published by the Association of Official Anylitical
Chemists, Washington, D. C.
Inquiries regarding this circular should refer to its number and be addressed to your
Regional Director, Bureau of Alcohol, Tobacco and Firearms.
Rex D. Davis